DNA Nanostructures for High-Throughput Cryo-EM Studies of Small Macromolecules Single particle cryo-electron microscopy (cryo-EM) is an approach for visualizing structures of macromolecules and their complexes at near-native conditions without the need for large sample quantities or removal of flexible regions often required for alternative techniques such as X-ray crystallography. Recent improvements in microscope hardware and data-processing software have helped to achieve near-atomic structure determination of macromolecules by cryo-EM allowing, for example, the visualization of individual amino acid side chains of protein targets. However, cryo-EM is quite limited for structure determination of small (<100 kDa) macromolecules. Cryo-EM image data are low contrast, and small particles often lack well-defined structural features required for the image alignment step of 3D reconstruction. Additionally, the method is technically challenging, low-throughput, and expensive?further hindering its widespread adoption. We propose to use DNA nanotechnology to develop a novel suite of tools to overcome the size and throughput limitations of cryo-EM. DNA nanotechnology allows us to create soluble nanostructures with an unprecedented combination of spatial resolution and chemical versatility. In principle, we can attach any moiety to our devices as long as it can be coupled to DNA, or to a DNA-binding molecule. We can build structures with dimensions ranging from 10 nanometers to 1 micrometer in size, but still create structures in which the location of every atom is defined with atomic or near-atomic resolution. We will design and optimize megadalton-sized DNA ?hinge? nanostructures that will bind and orient small macromolecules and serve as high-contrast fiducial markers for cryo-EM imaging and tomography. We will also construct DNA ?barcode? nanostructures and attach them to the DNA hinges for sample multiplexing. We will validate our methods by determining the structure of a well-characterized DNA-binding protein that has been previously crystalized. We will then work with collaborators to study several macromolecules of unknown structure. This technology will hugely improve our ability to solve near-atomic resolution cryo-EM structures of small macromolecules in a high-throughput manner. We will apply our method to study macromolecules with relevance to several human diseases, and expect that our efforts will ultimately enhance structure-based drug design efforts to combat those diseases.